Chronic opioid receptor activation frequently leads to the sensitization of adenylyl cyclase to stimulators after the inhibitory agonist has been removed (AC superactivation). AC superactivation after chronic opioid agonist exposure is thought to contribute to the development of opioid tolerance, dependence, and withdrawal (Williams et al., 2001). A better understanding of the molecular mechanisms of chronic δ-opioid agonist treatment-mediated AC superactivation should aid in the development of longer acting analgesics with fewer side effects.

We have reported previously that in Chinese hamster ovary (CHO) cells stably expressing the human δ-opioid receptor (hDOR/CHO), chronic δ-opioid agonist treatment gives rise to AC superactivation (Malatynska et al., 1996). In addition, we also demonstrated that chronic δ-opioid agonist treatment of the hDOR/CHO cells augments 32P incorporation into proteins immunoreactive with an AC V/VI-specific antibody (Varga et al., 1999). The identity of protein kinase(s) involved in chronic δ-opioid agonist-mediated phosphorylation in hDOR/CHO cells, and the role of the phosphorylation in AC superactivation, however, have not been investigated previously. In the present work, we studied the effect of protein kinase inhibitors on chronic δ-opioid agonist-mediated AC superactivation.

Recent data (Tan et al., 2001) indicate the important role of the protein kinase p74Raf-1 in phosphorylation of adenylyl cyclase VI in transfected human embryonic kidney 293 cells. Moreover, Raf-1-mediated phosphorylation led to the sensitization of AC VI to subsequent stimulation by forskolin or Gsα. The investigators (Tan et al., 2001) have also demonstrated that a dominant negative Raf-1 construct (NΔRaf) attenuates both phosphorylation and sensitization of AC VI. Interestingly, we found previously that AC VI is the major adenylyl cyclase isoenzyme in CHO cells and that a selective inhibitor of Raf-1 (GW5074) significantly attenuates chronic deltorphin II treatment-mediated adenylyl cyclase superactivation in hDOR/CHO cells (Varga et al., 2002).

Materials and Methods

Chemicals. Calmidazolium was purchased from Calbiochem (San Diego, CA), genistein was from Tocris Cookson, Inc. (Ellisville, MO), and chelerythrine and 3-(3,5-dibromo-4-hydroxybenzylidene-5-iodo-1,3-dihydro-indol-2-one (GW5074) were from Sigma-Aldrich (St. Louis, MO). (+)-4-[(αR)-α-((2S,5R)-4-Allyl-2,5-dimethyl-1-piperazinyl)-3-methoxy-benzyl]-N,N-diethyl benzamide (SNC 80) was synthesized at the National Institutes of Health (Bethesda, MD), in the laboratory of Kenner C. Rice. [d-Pen2-d-Pen5]-enkephalin (DPDPE) and deltorphin II ([d-Ala2]deltorphin II) were synthesized at the University of Arizona (Tucson, AZ), in the laboratory of Victor J Hruby. [32P]Orthophosphate (3000 Ci/mmol) was purchased from PerkinElmer Life Sciences (Boston, MA). All other compounds were obtained from commercial sources.

Measurement of Forskolin-Stimulated cAMP Formation. After chronic drug treatment, the cells were washed three times (15 min each) with fresh IMDM. The IMDM was then aspirated and replaced with 5 mM 3-isobutyl-1-methylxanthine (Sigma-Aldrich) in IMDM. Adenylyl cyclase was stimulated with 100 μM water-soluble forskolin (7-deacetyl-7-(O-N-methylpiperazino)-γ-butyryl, diHCl) (Calbiochem). The cells were incubated in a humidified incubator at 37°C (5% CO2), for 20 min. The reaction was terminated by replacing the medium with ice-cold Tris-EDTA buffer (50 mM Tris-HCl, 4 mM EDTA, pH 7.5). The cells were lysed by boiling (10 min) and centrifuged. Then 50 μl of the supernatant was incubated with 4 nM [3H]cAMP (PerkinElmer Life Sciences) and 30 μg/ml protein kinase A (Sigma-Aldrich). Serial dilutions of cAMP were run in parallel to obtain a cAMP standard curve. After a 2-h incubation at 4°C, activated charcoal (26 mg/ml) (NORIT, Amersfoort, The Netherlands) was added to adsorb free cAMP. The mixture was then centrifuged and 200 μl of the supernatant was counted in EcoLite (ICN Pharmaceuticals, Costa Mesa, CA) scintillation fluid.

Phosphorylation of Adenylyl Cyclase VI in the hDOR/CHO Cells after Chronic δ-Opioid Agonist (SNC 80) Treatment. A previously described (Varga et al., 1999) metabolic labeling/immunoprecipitation method was used to measure 32P incorporation into the protein band immunoreactive with an ACV/VI-specific antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA). Briefly, hDOR/CHO cells were phosphate-starved and labeled with [32P]orthophosphate. In experiments where specific protein kinase inhibitors were used, the inhibitors were added together with the [32P]orthophosphate. After a 1-h preincubation, SNC 80 (10 nM–1 μM) was added and the incubation continued for 1–24 h. After the agonist treatment, the cells were thoroughly washed and homogenized in solubilization buffer (50 mM Tris, 250 mM sucrose, 1 mM EDTA, 5 mM MgCl2, 1 mM dithiothreitol, 50 mM NaF, 10 mM Na-pyrophosphate, and 10 μl/ml protease inhibitor cocktail, 0.2 mM Na-orthovanadate, and 100 nM okadaic acid, pH 7.4). The lysate was centrifuged, precleared by incubating in the presence of 1 μg of preimmune rabbit IgG, and 10 μl of protein A-agarose for 1 h. After centrifugation, the precleared lysate was incubated with the ACV/VI-specific antibody and protein A-agarose beads for 4 h. After thorough washes in solubilization buffer, with detergent concentrations reduced to 0.075% Triton X-100, 0.05% Igepal CA-630 and 0.1% digitonin (4°C), the immuno-complexes, were eluted in glycine-Cl buffer (pH 2.3), neutralized, solubilized in Laemmli buffer, and resolved on 7.5% SDS-PAGE. The gel was silver-stained, dried, and subjected to autoradiography. The amounts of immunoprecipitated protein, and the extent of 32P incorporation, were quantified by scanning densitometry of the silver stained gel and the autoradiography film, respectively.

Phosphorylation of Adenylyl Cyclase VI in the hDOR/CHO Cells upon Chronic δ-Opioid Agonist (SNC 80) Treatment. It was shown previously that chronic opioid treatment-mediated AC superactivation is isoenzyme-specific (Avidor-Reiss et al., 1997). AC VI was one of the few AC isoenzymes that became sensitized to forskolin stimulation upon chronic opioid receptor stimulation (Avidor-Reiss et al., 1997). We have previously determined that AC VI is a major native isoenzyme in CHO cells (Varga et al., 1998). Thus, we hypothesized that AC VI is that major isoenzyme involved in forskolin-stimulated cAMP overshoot upon chronic δ-opioid agonist treatment in the hDOR/CHO cells.

Because the catalytic activity of adenylyl cyclase isoenzymes is frequently regulated by protein kinases (Ishikawa, 1998), we tested the effect of chronic δ-opioid agonist treatment on the phosphorylation state of AC VI in hDOR/CHO cells. A metabolic labeling/immunoprecipitation method, followed by SDS-PAGE, was used to measure 32P incorporation into AC VI upon chronic SNC 80 treatment, as described previously (Varga et al., 1999). Two bands, with apparent molecular masses of about 130 and 200 kDa, were routinely obtained on both the silver-stained gel and the autoradiography film. Figure 2 shows a representative autoradiography film obtained after SNC 80 treatment of hDOR/CHO cells for 4 h with increasing doses of the drug. The 200-kDa band is presumably the glycosylated form of the 130-kDa protein, because only the 130-kDa band was apparent on the gel when after the immunoprecipitate was pretreated with N-aminoglycosidase F (data not shown). Immunoprecipitation was prevented by saturation of the antibody with a blocking peptide (Santa Cruz Biotechnology, Inc.). The optical densities of the bands in the silver-stained gel (protein content) and in the autoradiography film (32P incorporation) were measured by scanning densitometry. Because a similar protein/phosphate ratio was measured for the 130- and the 200-kDa bands in initial experiments, we routinely used the 200-kDa band to quantitate phosphorylation. Data in Figs. 2 and 3 show the density ratios (32P incorporation/protein stain) calculated as the percentage of the IMDM-treated control. Chronic (4-h) SNC 80 treatment-mediated phosphorylation of AC VI was at a maximal level (557% of untreated control, range 400–714, n = 2) at 100 nM SNC 80 concentrations (Fig. 2). Figure 3 shows a time course for SNC 80 (1 μM)-mediated 32P incorporation into the 200-kDa band. Phosphorylation was maximal (303 ± 61% of control, p < 0.001, n = 7) after 4-h treatment. Phosphorylation of AC VI remained at similar levels upon longer (up to 24 h, 204 ± 29%, p < 0.01, n = 3) treatment.

Dose-response of chronic (4 h) SNC 80-mediated phosphorylation of AC VI: hDOR/CHO cells were metabolically labeled with 200 μCi/ml [32P]orthophosphate and treated with increasing concentrations of SNC 80 for 4 h. Cell lysates were immunoprecipitated by an AC V/VI-specific antibody and protein A-agarose. The immunoprecipitated proteins were resolved on 7.5% SDS-PAGE. The gels were silver-stained and subjected to autoradiography. The figure shows a representative autoradiogram. The locations of the molecular weight standards on the corresponding silver-stained gel are indicated by arrows. Optical densities in the silver-stained gel (protein content) and in the autoradiography film (32P incorporation) were measured for the 200-kDa band using scanning densitometry. 32P incorporation was normalized for the amount of protein by calculating a ratio between the measured optical densities. The values underneath each SNC 80 dose show the range and the mean of the normalized 32P incorporation, calculated as percentage of IMDM-treated control (n = 2).

Time course of SNC 80-mediated phosphorylation of AC VI: hDOR/CHO cells were metabolically labeled with 200 μCi/ml [32P]orthophosphate before treatment with SNC 80 (1 μM) for different time intervals. The cell lysates were immunoprecipitated, and the immunoreactive proteins were measured as described in the legend for Fig. 2. The density ratio (32P incorporation/protein) increased to 303 ± 61% of IMDM-treated control (n = 7) after 4 h SNC 80 pretreatment, and remained at a similar level upon longer (up to 24 h, 204 ± 29%, n = 3) treatment.

Discussion

Chronic stimulation of Gi/o protein-coupled receptors often leads to a compensatory increase in the response of adenylyl cyclase to stimulators (AC superactivation) after the removal of the inhibitory agonist. AC superactivation was first described for the opioid receptors in NG108-15 cells (Sharma et al., 1975). Although AC superactivation is thought to play an important role in opioid tolerance, dependence, and withdrawal, its molecular mechanism is still not completely understood (Williams et al., 2001). Down-regulation of the opioid receptors is probably not involved in AC superactivation because increased cAMP levels (cAMP overshoot) become evident after agonist withdrawal. Down-regulation of Gi/o proteins (vanVliet et al., 1991), subcellular translocation of Gi (Bayewitch et al., 2000) and Gs (Ammer and Schulz, 1997) proteins, attenuation of phosphodiesterase activity (Law and Loh, 1993), and switching from Gi/o-mediated signaling to Gs-mediated signaling (Crain and Shen, 1998) have been suggested as possible explanations for cAMP overshoot.

AC superactivation has been observed in brain areas involved in opiate addiction, but it can also be demonstrated in recombinant cell lines heterologously expressing opioid receptors. We (Malatynska et al., 1996), and others (Avidor-Reiss et al., 1995) demonstrated that chronic opioid treatment of CHO cells expressing δ-or μ-opioid receptors leads to AC superactivation. It has also been shown previously that adenylyl cyclase superactivation is isoenzyme-specific (Thomas and Hoffman, 1996; Avidor-Reiss et al., 1997, Cumbay and Watts, 2001). Using a reverse transcription-polymerase chain reaction method, we previously found that AC VI and AC VII are the major AC isoenzymes expressed in CHO cells (Varga et al., 1998). It was demonstrated previously that AC VI is one of the few AC isoenzymes that becomes sensitized to forskolin after chronic opioid receptor stimulation and subsequent withdrawal (Avidor-Reiss et al., 1997). Therefore, we hypothesized that the AC VI isoenzyme has an important role in augmentation of forskolin-stimulated cAMP formation by chronic δ-opioid agonist treatment in hDOR/CHO cells.

Compensatory feedback regulation of the concentration or/and catalytic activity of adenylyl cyclase is an attractive hypothesis to account for cAMP overshoot. Thus, it was shown previously that chronic opioid treatment increases the mRNA levels of AC isoenzymes in the nucleus accumbens (Nestler and Aghajanian, 1997) and AC VII in the ileum longitudinal muscle myenteric plexus (LMMP) (Rivera and Gintzler, 1998). The relatively rapid kinetics and cycloheximide insensitivity of AC superactivation in hDOR/CHO cells (Avidor-Reiss et al., 1995; Rubenzik, 2002), however, suggests that new protein synthesis is not the sole mechanism involved in this process. It was also demonstrated previously that G protein βγ-subunits, released upon agonist-stimulation of the transiently transfected μ-opioid receptor, have a major role in AC superactivation in COS-7 cells (Avidor-Reiss et al., 1996). Previously, we also found that overexpression of a free βγ-subunit scavenger (the α-subunit of rod transducin, αt1) completely attenuates SNC 80-mediated AC superactivation in hDOR/CHO cells (Rubenzik et al., 2001).

Because phosphorylation regulates the catalytic activity of adenylyl cyclase isoenzymes (Ishikawa, 1998), previously we tested whether chronic δ-opioid agonist treatment leads to phosphorylation of the AC VI isoenzyme in hDOR/CHO cells. We found that concurrent to AC superactivation, chronic SNC 80 treatment augments 32P incorporation into a 130- and a 200-kDa protein band, immunoprecipitated by an AC V/VI-specific antibody from hDOR/CHO cells. Because only the 130-kDa band was apparent after aminoglycosidase F treatment of the hDOR/CHO cell extracts, the 200-kDa band presumably corresponds to the glycosylated form of the 130-kDa protein. Chronic SNC 80-mediated 32P incorporation in the hDOR/CHO cells was attenuated by naltrindole, a selective δ opioid receptor antagonist (Varga et al., 1999) and was SNC 80 dose- and treatment time-dependent (this work). Importantly, the time course of the onset of SNC 80-mediated AC superactivation (Rubenzik, 2002) and AC phosphorylation (this work) are remarkably similar. Interestingly, Chakrabarti et al. (1998) simultaneously observed that chronic morphine treatment augments AC phosphorylation in LMMP preparations.

Chakrabarti et al. (1998) previously demonstrated that chronic morphine treatment-mediated phosphorylation of adenylyl cyclase in guinea pig LMMP preparation was attenuated by chelerythrine pretreatment, indicating the involvement of PKC in this process. Agonist-activated opioid receptors in recombinant CHO cells have been shown to stimulate IP1 formation (Rubenzik et al., 2001), arachidonate release, and phosphatidylinositol-3 kinase (Fukuda et al., 1996). Either of these pathways may lead to the activation of PKC isoenzymes in hDOR/CHO cells. However, because we found that the PKC inhibitor chelerythrine has only a very moderate effect on AC superactivation, PKC-mediated phosphorylation of the adenylyl cyclase isoenzymes is unlikely to play a major role in chronic δ-opioid agonist-mediated AC superactivation in hDOR/CHO cells.

Recently, phosphorylation of AC VI by the Raf-1 protein kinase has been demonstrated (Tan et al., 2001). Interestingly, similar to chronic opioid treatment-mediated AC superactivation, Raf-1-mediated phosphorylation was shown to sensitize the AC VI to stimulators, such as forskolin and Gsα. Therefore, we tested the involvement of Raf-1 in chronic δ-opioid agonist-mediated AC superactivation in hDOR/CHO cells. We found that pretreatment of hDOR/CHO cells with the selective Raf-1 inhibitor GW5074 significantly attenuates chronic δ-opioid agonist-mediated AC superactivation (Varga et al., 2002).

Previously, it was concluded that the mitogen-activated protein kinase pathway is not involved in AC superactivation, because neither overexpression of dominant negative Ras nor the phosphatidylinositol-3-kinase inhibitor wortmannin was able to attenuate AC superactivation in COS-7 cells cotransfected with the μ-opioid receptor and AC V (Avidor-Reiss et al., 1996). However, because the catalytic activity of Raf-1 is modulated by multiple mechanisms, blocking a single pathway may only shunt the signal to alternative, parallel pathways. Thus, simultaneous inhibition of both ras-dependent- and ras-independent pathways may be necessary to achieve complete inhibition of AC superactivation, as we indeed found in hDOR/CHO cells.

We also found that both AC superactivation and AC VI phosphorylation can be completely attenuated by pretreatment with calmidazolium. Calmidazolium is a calmodulin antagonist that competes with calmodulin-sensitive intracellular effectors in low micromolar concentrations (Gietzen et al., 1981). Interestingly, the involvement of a calmodulin-sensitive step, upstream of opioid-mediated transactivation of receptor- and nonreceptor tyrosine kinases and the small G protein Ras, was previously demonstrated in opioid receptor-mediated MAPK activation (Belcheva et al., 2001). In addition, recent data indicate that calmidazolium also inhibits Raf-1 directly, by antagonizing calmodulin binding to the enzyme (Egea et al., 2000). It should be noted, however, that maximal values for forskolin-stimulated cAMP formation were also different in IMDM- and calmidazolium-treated hDOR/CHO cells. Because we previously found that CHO cells do not express calmodulin- or calmodulin kinase-sensitive AC isoforms the reason for increased basal cAMP formation in calmidazolium-treated cells is presently not clear. Calmidazolium, however, is a nonselective calmodulin antagonist that interferes with a number of other intracellular enzymes, such as calmodulin-dependent protein phosphatases and phosphodiesterases. This may change the basal phosphorylation of every AC isoenzyme in CHO cells and also may affect cellular cAMP degradation rate, contributing to increased basal cAMP formation in calmidazolium-treated cells.

In summary, we have demonstrated that chronic SNC 80 treatment augments 32P incorporation into two protein bands immunoreactive with the ACV/VI specific antibody. Both chronic SNC 80-mediated 32P incorporation and AC superactivation are naltrindole-sensitive and exhibit similar SNC 80 dose and time dependence. Calmidazolium and a selective Raf-1 inhibitor (GW5074) significantly attenuated chronic δ-opioid agonist-mediated adenylyl cyclase superactivation in hDOR/CHO cells. Tyrosine kinase (genistein) and PKC (chelerythrine) inhibitors individually had a minimal effect. However, simultaneous treatment with both genistein and chelerythrine abolished AC superactivation. Based on our experimental data, we suggest that multiple redundant pathways contribute to δ-opioid receptor-mediated activation of Raf-1, that in turn leads to the phosphorylation and sensitization of AC VI in hDOR/CHO cells. Figure 6 shows a putative molecular model to interpret the role of chelerythrine-, genistein-, and calmidazolium-sensitive signal transduction pathways in Raf-1 activation and AC VI phosphorylation. Adenylyl cyclase superactivation is an important molecular mechanism contributing to the development of tolerance and dependence to chronic opioid treatment. Better understanding of this important cellular compensatory mechanism should ultimately lead to the development of longer acting analgesic drugs with fewer side effects.

Putative molecular mechanism of δ-opioid agonist-mediated phosphorylation of adenylyl cyclase VI in hDOR/CHO cells. Stimulation of the hDOR by an opioid agonist (*) in CHO cells liberates G protein βγ subunits. Free G protein βγ subunits interact with multiple effectors, leading to the activation of PKC isoforms; recruitment of β-arrestin (β-arr) and the nonreceptor tyrosine kinase Src; and transactivation of tyrosine kinase receptors (RTK), such as the platelet-derived growth factor receptor. Activation of PKC and Src, on the other hand, results in Raf-1 activation through Ras-independent- or Ras-dependent pathways, respectively. Activated Raf-1 phosphorylates and sensitizes adenylyl cyclase VI (AC VI), leading to a cAMP overshoot. The putative molecular targets of the inhibitors used in the present work are indicated in the figure. Calmidazolium is a calmodulin antagonist, genistein is a tyrosine kinase inhibitor, whereas chelerythrine nonselectively inhibits PKC isoenzymes. GW5074 is a selective Raf-1 inhibitor. MEK, mitogen-activated protein kinase kinase.

Acknowledgments

We thank Carol Haussler and Michelle Thacher for the maintenance of the hDOR/CHO cell culture.

Footnotes

This work was supported in part by grants from the National Institutes of Health and the Arizona Disease Control Research Commission.